ADVERTISEMENTS:
The following points highlight the top four types of direct gene transfer in plants. The types are: 1. Plant Cell Transformation by Ultra-Sonication 2. Liposome Mediated Gene Transfer 3. Electroporation 4. Particle Bombardment Gun.
Type # 1. Plant Cell Transformation by Ultra-Sonication:
The Biotechnology Research Centre, Beijing (China) has used technique of ultra-sonication for gene transfer in plants such as wheat, tobacco and sugarbeet. When the cultured explants were sonicated with plasmid DNA carrying marker genes such as cat, npt II and gus and sonicated cells transferred to selective medium, shoots have grown successfully.
Type # 2. Liposome Mediated Gene Transfer:
Liposomes are microscopically small-sized lipid bags. They are bound by phospholipid bilayers and contain aqueous chamber which may contain water soluble molecules such as plasmids or foreign DNA. By using polyethylene glycol (PEG), plasmid containing liposomes may be stimulated to fuse with protoplast.
ADVERTISEMENTS:
In several plants such as carrot, tobacco, petunia, etc., this technique has been used for successful transfer of genes. Due to endocytosis of liposomes, DNA/plasmid enter, the protoplasts.
This technique has the following advantages:
(1) Low toxicity;
(2) Protection of DNA/RNA from nucleases that lyse them;
ADVERTISEMENTS:
(3) Long stable storage of DNA fragments in liposome;
ADVERTISEMENTS:
(4) Applicability in various cell types, and
(5) High level of re-productivity.
Transfection by liposomes in mice has given good results.
Type # 3. Electroporation:
Eletroporation is now used to transfer the foreign DNA into the fragile cells. Brief pulses of high voltage electricity (about 350 V) is applied to protoplast suspension containing naked or recombinant plasmids. The electric pulses induce the formation of large pores in plasma membrane.
These pores give a passage through which the foreign DNA can enter into the protoplasts and thus, increase in transformation frequency. The transformed protoplasts are cultured for about one month. These develop the microcalli which are plated on solid medium containing selective marker (e.g., kanamycin).
After 37-45 days, the calli are analysed for the presence of differences in transformed cells. By using electroporation method, genes of choice have been successfully transferred in protoplasts of wheat, rice, petunia, sorghum, maize and tobacco.
Type # 4. Particle Bombardment Gun:
It was developed by Prof. Stanford and co-workers at Cornell University (USA) in 1987. As the term denotes, it shoots foreign DNA into plant cells or tissues at a very high speed. This technique is also known as particle gun method, biolistic process, gene gun and micro-projectile gun.
ADVERTISEMENTS:
This technique is most suitable for those plants which hardly regenerate and do not show sufficient response to gene transfer through Agrobacterium, for example, rice, wheat, com, sorghum, chicken pea and pigeon pea.
Recently, transfection has been successful in both mitochondria and chloroplasts (both have been proven to be difficult targets for genetic engineering because they have double-membrane walls) using a biolistic process.
The gene gun consists of a chamber connected to an outlet to create vacuum. At the top, a cylinder is temporarily sealed off from the rest of chamber with a plastic rupture disk. Helium gas flows into the cylinder.
A plastic microcarrier is placed close to rupture disk. It contains DNA- coated tungsten particle, the microscopic pellets (i.e., coated micro-projectiles). While work the apparatus is placed in Laminar flow just to maintain sterile conditions. The target cells/tissues are placed in the apparatus.
ADVERTISEMENTS:
A stopping screen is placed between the target cells and microcarrier assembly. Helium gas is flown in the cylinder at high velocity. When pressure of cylinder exceeds the bursting point of plastic disk, it gets ruptured. Helium shock waves propel the plastic microcarrier containing DNA-coated micropellets.
The stopping screen allows the micropellets to pass through and deliver DNA into target cells. The transformed cells are regenerated onto nutrient medium. The regenerated plant tissues are selected over culture media containing either antibiotic or herbicides. The selected plants are then analysed for expression of foreign DNA.
Using gene gun, scientists have got success in delivering forcing DNA into epidermal tissue of Allium cepa, scutellar tissue of maize and leaf and cell cultures of many crops. In addition to bacterial cells, algae, fungi, plants organelles (e.g., chloroplast and mitochondria), animal or human cells and fruitfly embryos have been successfully transformed.
Ramaiah and Skinner (1997) produced transgenic alfalfa through direct delivery of DNA into pollen grains by particle bombardment method. In 1998, scientists of Plant Transformation Group at ICGEB (New Delhi) got success in transforming human interferon gamma gene into chloroplasts of tobacco, maize, etc. using a particle gun mediated gene transformation.
